PROJECT SUMMARY: We propose to investigate a novel epitope mapping approach of SARS-CoV S proteins, using RNA phage Q? displayed peptides, and to evaluate the potential of these engineered hybrid phages as antibody neutralizing determinants critically important in the development of an efficacious vaccine candidate. Among the proteins coded by the SARS-CoV genome, S protein mediates its cell entry and induces neutralizing antibodies. These pivotal functions are achieved by key amino acid peptide(s) through their exposed position on the virus surface. Phage Q? is a small positive strand RNA virus with a 4.2 kb genome encoding 4 proteins. These are coat protein (Cp), maturation (or A2) protein, read-through or minor coat protein (or A1), and the RNA-dependent RNA polymerase or RNA replicase (RdRp) protein. As an RNA virus, phage Q? possesses a key feature for its rapid evolution and adaptation: the RdRp protein that does not have proofreading activity during replication, resulting in higher mutation rates which simulate in vitro evolution and affinity-maturation processes. The A1 protein has recently been successfully used for fusion and display of randomized peptides in our laboratory, which is important because of its number and position on the phage surface. These fundamental concepts of RNA display will be exploited to investigate the following three specific aims: 1. Design, construct, express and characterize hybrid phages Q? exposing peptide of S protein epitopes on the exterior surface. Potential S protein continuous and discontinuous (chimeric) epitopes will be localized and checked for exposition on A1 protein, using a combination of three computer bioinformatics software. The identified epitopes will be designed as oligonucleotides and cloned by fusion to the end of A1 minor coat protein gene for the novel RNA display system. These constructs will be characterized and expressed for hybrid phages (phagotopes) production. For any testing and selection of variant phages, we will use anti-S Abs. 2. Randomize and optimize the major epitopes of S protein. A novel biopanning method will be developed for selecting the appropriate randomized phages mimicking S protein epitopes (mimotopes) against the existing SARS-CoV and S protein mono/polyclonal antibodies. The selected mimotope(s) will be easily optimized and evolved through at least ten rounds of biopanning. The determinants of the randomized mimotopes pool will also be classified by antibody type, and studied for any potential affinity to other viral cellular receptors different from the natural SARS-CoV. Non-selected hybrid phages will be analyzed in correlation with the antibodies. 3. Stabilize and initiate evaluation of the potential binding and neutralization of variant phages to SARS- CoV antibodies. We hypothesize that epitope peptide flanked by paired cysteines can be stabilized on the surface of the mimotopes and/or phagotopes. The prepared hybrid phages will be analyzed and evaluated for their potential binding to all SARS-CoV antibodies and/or sera. The antibodies will be tested and classified for affinity with the phagotopes and/or mimotopes in comparison to the wild type SARS-CoV or pseudotype model.